The overall objective of this research is to understand the process of N-glycosylation that occurs in concert with protein translocation. The enzyme oligosaccharyl transferase (OT) catalyzes the N-glycosylation of proteins in all eukaryotic cells. N-linked glycoproteins play essential roles in cell growth, communication, development and many other cellular functions. Additionally, the N-linked glycans of many infectious agents (viruses, fungi, and parasites) mediate the ability of these organisms to infect humans. N-linked glycan chain/s of many soluble and membrane glycoproteins participate in an important quality control step required for their successful synthesis and export from the endoplasmic reticulum; a large number of different congenital disorders (CDGS) are found to be associated with improper glycosylation and folding. As a result, the OT enzyme may ultimately be an important target to regulate these processes to allow physicians to treat infectious diseases, congenital disorders and abnormal tissue processes such as cancer. Studies will be carried out in yeast. All nine subunits of OT are transmembrane proteins in the endoplasmic reticulum and they have been cloned and sequenced. The available evidence indicates that all 9 proteins exist in a complex. The first specific aim is to test the role of the individual OT subunits in the N-glycosylation reaction based on current biochemical and genetic information. This will be achieved by use of diverse genetic, biochemical and biophysical techniques. An optimized assay for OT activity will be developed to accomplish specific aims 1 and 2. A particular emphasis will be placed on identifying the subunit(s) that recognizes Dol-P-P-oligosaccharide as a donor of the oligosaccharide chain. The second specific aim will be to study the physical structure of the purified soluble form of OT by cryo electron microscopy techniques and the application of antibody directed to the various subunits. To refine our level of understanding of the structure of OT, the purified OT complex will be reconstituted in phospholipid liposomes in order to determine its structure of 2D crystals by electron crystallography. The third specific aim will be to elucidate the site(s) of interaction of the OT subunit SttSp, the subunit believed to contain the active site of OT with the translocon component Sec61p. This will be achieved using biochemical and genetic approaches. Thus the catalytic mechanism of the OT reaction in the first specific aim and structural features of the OT complex in the second specific aim will be addressed. In a physiological milieu, the two essential processes of N- glycosylation and protein translocation are concomitant events. Understanding the molecular basis of the concerted action of these two processes will be the goal of the third specific aim. [unreadable] [unreadable] [unreadable]